All living cells possess the means to adapt to their environments. Cells rely, to a great extent, on extracellular molecules as a means by which to receive stimuli from their immediate environment. These extracellular signals are essential for the correct regulation of such diverse cellular processes as differentiation, contractility, secretion, cell growth, cell migration, contact inhibition and apoptosis. The external environmental signals received by the cell are transduced into the cell via activation of membrane-situated receptors. Activation of cell surface receptors is often dependent upon protein-protein interactions, including receptor-ligand binding, and receptor dimerization (homodimerization or heterodimerization) or oligomerization. Aberrant signalling can disrupt any of these cellular processes with detrimental results.
For reviews of signal transduction pathways see, e.g., Campbell, 1997, J. Pediat. 131:542-544; Hamilton, 1997, J. Leukoc. Biol. 62:145-155; Soede-Bobok & Touw, 1997, J. Mol. Med. 75:470-477; Heldin, 1995, Cell 80:213-223; Kishimoto et al. 1994, Cell 76:253-262; Miyajima, et al. 1992, Annu. Rev. Immunol. 10:295-331; and Cantley, et al. 1991, Cell 64:281-302.
Protein-protein interactions also play an important role in processes concerning many other cellular and viral proteins and enzymes, in addition to cell surface receptors. In some instances disruption of protein-protein interactions will lead to the loss of polypeptide function. In certain instances, loss of function of an enzyme may be therapeutically desirable. For example, the protease from HIV is a dimer (McKeever et al. 1989, J. Biol. Chem. 264:1919-1921) and the dimerization of the protein is required for function (Guenet et al. 1989, Eur. J. Pharmacol. 172:443-451, and Babe et al. 1992, Protein Sci. 10:1244-1253). Some candidate molecules (Zhang et al. 1991, J. Biol. Chem. 266:15591-15594 and Schramm et al. 1993, Biochem. Biophys. Res. Commun. 194:595-600) that block HIV protease function do so by disrupting dimerization.
Given the ubiquitous and important nature of protein-protein interactions in signal transduction pathways and in other cellular processes, compounds by which such interactions can be modulated would be very advantageous.
Attempts to identify ways to modulate such events have been reported. See, e.g., chimeric receptor studies reported by Schlessinger, and the chimeric signal transduction systems reported by Menzel et al. (utilizing heterologous Vibrio cholerae toxR in E. coli; U.S. Pat. No. 5,521,066), Utsumi (utilizing a chimeric E. coli Tar protein; Utsumi et al. 1989, Science 245:1246-1249), Riedel (utilizing different eukaryotic hormone receptors in tissue culture; Riedel et al. 1989, EMBO J. 8:2943-2954) and Moe (utilizing a bacterial aspartate binding domain and the insulin receptor (Moe et al. 1989, Proc. Natl. Acad. Sci. USA 5 86:5683-5687). See also King et al. (U.S. Pat. Nos. 5,482,835 and 5,739,029; methods for screening for agonists and antagonists for G-protein coupled receptors). Each of these methods, however, exhibits significant limitations in either specificity, broad versatility and/or sensitivity.
Despite such reports, therefore, as yet, no efficient, sensitive, versatile high throughput procaryote-based system has yet been described for identifying protein-protein interactions or for identifying compositions for modulating such interactions.